Name: bronchoalveolar lavage exosomes in lipopolysaccharide-induced septic lung injury
Uploaded: 2018-05-21T15:00:00
Description: Watch this Scientific Journal Video about Bronchoalveolar Lavage Exosomes in Lipopolysaccharide-induced Septic Lung Injury at JoVE.com

The authors have declared no conflict of interest.

The overall protocol requires 2 days, including the first day of sepsis induction and isolation of BAL fluid from the animal, and the second day for exosomes isolation from the mouse BALF. All procedures have been reviewed and approved by the Institutional Animal Care and Use Committee at Atlanta VA medical center.
1. Mouse Acute Septic Lung Injury Model
<ol>
	<li>Use 6 - 8 weeks- old male wild-type C57BL/6J mice (weight 20 - 22 g) for the animal model, and perform all procedures using asept...

<ol>
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Mouse models of diseases are commonly used to evaluate the physiological function of specific genes and to reduce the cost of experimentation2. The acute septic lung injury described here mimics inflammatory response seen in humans with ARDS. This model is relevant to investigate the molecular pathogenesis, development of biomarkers and to test potential new therapies5.
Co-culture systems are relevant for in vivo studies in the context o...

ALI and ARDS are the life-threatening forms of respiratory failure with severe hypoxemia caused by non-cardiogenic pulmonary edema which affects approximately 1 million people worldwide annually1. The etiology of ARDS includes direct injury to the lungs from infections or aspiration and a variety of indirect insults. Over the last decade there has been an increased understanding of the molecular pathogenesis of ARDS, however, specific targeted treatments for ARDS are yet to be developed2,3.
Several animal models of acute lung injury have been developed which provide a bridge for translating experimental therapies to human studies4,5. Commonly used models include local installation of oleic acid, bacteria, LPS, and bleomycin. Other approaches include ischemia-reperfusion, cecal ligation puncture, mechanical ventilation-induced stretch injury, hyperoxia or systemic administration of bacteria and LPS5. These models provide a useful biological system to test clinical hypotheses and for the development of potential therapies. To simulate human ARDS, animal models should reproduce inflammation and acute injury to the epithelial and endothelial cells with defects in barrier function in the lungs.
Exosomes are membrane vesicles with 20 - 200 nm in diameter, whose molecular content contains proteins, DNA, RNA, and lipids, and facilitate inter-cellular communications in tissue microenvironment via molecular composition transfer. Exosomes are secreted by multiple types of cells, such as endothelial cells, epithelial cells, smooth muscle cells and tumor cells, and exist in human body fluid. Studies indicate that exosomes regulate cross-talk between immune cells and stromal cells during infectious and sterile inflammatory diseases, and their abnormal release appears to be regulated by various natural and experimental stimuli during physiological and pathological processes6. Such communication network may play an important role in the pathogenesis of lung diseases and may affect pathophysiological progression7,8. As 18 - 22 nucleotide non-coding RNAs, miRNAs exist in both tissue and body fluids, plasma, sera, and modulate mRNA expression at post-translational level9,10.
Packaged miRNAs in exosomes influence differentiation and function of multiple types of cells, and excessive levels are associated with a variety of diseases, including cancer, lung diseases, obesity, diabetes, and cardiovascular disease11,12,13,14,15,16. Entry into the recipient cells and shuttling of exosomal miRNAs facilitate intercellular communications modifying the hemostasis of microenvironment17,18. Acute lung-injury is a complex processes, involving multiple cell types with extensive intercellular communications through exosomes8. miR-155 and miR-146a share the common transcriptional regulatory mechanism and contribute to the inflammatory response and the immune tolerance19,20. Recent studies indicate that both modulate inflammatory response via exosomal miRNAs shuttling between immune cells21. However, the molecular mechanisms underlying modulatory effects of exosomal miRNAs on alveolar response to endotoxin remain unclear, undoubtedly the potential clinical relevance and translational implication merit further investigation.
Co-culture models are being employed to define the interaction of the specific cell types in the complex environment, such as inflammation and cancer22,23. These platforms provide an alternative strategy to interrogate cross talk between cell types particularly for immune and structural cells.
Intra-tracheal, aerosolized, intraperitoneal or systemic administration of LPS are widely used to induce experimental lung injury24,25,26, and have shown to induce epithelial and endothelial permeability defects. Here we use intraperitoneal LPS to induce a septic model of acute lung injury in mice. Within 24 h of intraperitoneal administration of LPS, permeability defects are induced in the lungs with recruitment of inflammatory cells. Further, we show that exosomes from BAL contain miRNA-155 and miR-146a, and exosomes from BAL fluid induce pro-inflammatory cytokines expression in recipient epithelial cells, including IL-6 and TNF-&#945;. These data are the first to show that exosomal miRNAs are secreted in BAL in this model of acute septic lung injury.

<table border="0" cellpadding="0" cellspacing="0" style="width:1276px;" width="1276">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:691px;">lipopolysaccharide</td>
			<td style="width:181px;">Sigma-Aldrich</td>
			<td style="width:224px;">Escherichia coli 055:B5</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PBS pH7.2(1X)</td>
			<td>Life technologies</td>
			<td>20012-027</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">mirVana miRNA isolation kit</td>
			<td>Thermo Fisher</td>
			<td align="right">449774</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TaqMan Gene Expression Master Mix</td>
			<td>Thermo Fisher</td>
			<td align="right">4369016</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">mmu-miR-155 (002571) primer</td>
			<td>Thermo Fisher</td>
			<td align="right">4427975</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">has-miR-146a (000468) primer</td>
			<td>Thermo Fisher</td>
			<td align="right">4427975</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">U6snRNA primer</td>
			<td>Thermo Fisher</td>
			<td align="right">4427975</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TNF-&#945;(Mm00443258_m1 ) primer</td>
			<td>Thermo Fisher</td>
			<td align="right">4331182</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">IL-6 (Mm00446190_m1&#160;&#160; )primer</td>
			<td>Thermo Fisher</td>
			<td align="right">4331182</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ZO-1 (Mm00493699_m1&#160; )primer</td>
			<td>Thermo Fisher</td>
			<td align="right">4331182</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">minimal essential medium (MEM)</td>
			<td>Thermo Fisher</td>
			<td>11095-072</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trysin-EDTA solution(0.05%)</td>
			<td>Thermo Fisher</td>
			<td align="right">25300054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Exosomes were labeled with PKH67 through PKH67 Green Fluorescent Cell linker Mini Kit&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>MINI67-1KT</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Exosome Spin Columns (MW3000)</td>
			<td>Thermo Fisher</td>
			<td align="right">4484449</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Beckman Coulter Optima L-100XP ultracentrifuge&#160;</td>
			<td>Beckman Coulter</td>
			<td>L-100XP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultra-clear centrifuge tubes</td>
			<td>Beckman Coulter</td>
			<td align="right">344058</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Applied Biosystems 7500 Fast Real-Time PCR System</td>
			<td>Thermo Fisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">transmission electron microscopes (TEM)</td>
			<td>JEOL Ltd</td>
			<td>120 kV TEM</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Olympus BX41 microscope</td>
			<td>Olympus</td>
			<td>BX41</td>
			<td></td>
		</tr>
	</tbody>
</table>

bronchoalveolar lavage exosomes in lipopolysaccharide-induced septic lung injury

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) represent a heterogeneous group of lung diseases which continues to have a high morbidity and mortality. The molecular pathogenesis of ALI is being better defined; however, because of the complex nature of the disease molecular therapies have yet to be developed. Here we use a lipopolysaccharide (LPS) induced mouse model of acute septic lung injury to delineate the role of exosomes in the inflammatory response. Using this model, we were able to show that mice that are exposed to intraperitoneal LPS secrete exosomes in Broncho-alveolar lavage (BAL) fluid from the lungs that are packaged with miRNA and cytokines which regulate inflammatory response. Further using a co-culture model system, we show that exosomes released from macrophages disrupt expression of tight junction proteins in bronchial epithelial cells. These results suggest that 1) cross talk between innate immune and structural cells through the exosomal shuttling contribute to the inflammatory response and disruption of the structural barrier and 2) targeting these miRNAs may provide a novel platform to treat ALI and ARDS.

To induce septic lung injury, mice were treated with intraperitoneal LPS (15 mg/kg). Within 24 h of LPS administration, neutrophilic influx was seen in the lungs, as shown in Figure 1A. Mouse BAL fluid was drawn, followed by isolation and purification of exosomes. Morphology of BALF exosomes was confirmed by electron transmission microscopy (Figure 1B).
Expression of se...

Watch this Scientific Journal Video about Bronchoalveolar Lavage Exosomes in Lipopolysaccharide-induced Septic Lung Injury at JoVE.com

Bronchoalveolar Lavage Exosomes in Lipopolysaccharide-induced Septic Lung Injury

Mice exposed to intraperitoneal LPS secrete exosomes in Broncho-alveolar lavage (BAL) fluid that are packaged with miRNAs. Using a co-culture system, we show that exosomes released in the BAL fluid disrupt expression of tight junction proteins in bronchial epithelial cells and increase expression of pro-inflammatory cytokines that accentuate lung injury.

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) represent a heterogeneous group of lung diseases which continues to have a high ...

The overall goal of this experiment is to delineate the role of exosomes in the inflammatory responses in an LPS induced, acute septic lung injury mouse model. So this method can help answer key questions related to cell cross talk in acute lung injury and acute respiratory distress syndrome. Especially intercellular communication within the alveolar-micro environment via exosome shuttling.The main advantage of this technique is that it allows the isolation and characterization of highly purified bronchoalveolar lavage exosomes derived from endotoxin treated septic mice with acute lung injury. Demonstrating the procedure is Dr.Zhihong Yuan and Dr.Chetna Bedi. To begin, load a one milliliter syringe equipped with a 27 gauge needle with 0.1 milliliters of LPS and PBS per mouse and manually restrain the first six to eight week old male C57 black 6J mouse.Deliver the LPS intra-peritoneally and return the animal to its cage. 24 hours later, make a five millimeter incision in the center of the neck of the first animal and use blunt forceps to gently separate the muscles to expose the trachea. Next, gently insert a 27 gauge needle attached to a sterile 10 milliliter syringe into the trachea and inject one milliliter of sterile PBS.Then immediately replace the 10 milliliter syringe with a three milliliter syringe equipped with a new 27 gauge needle. Aspirate the bronchoalveolar lavage fluid or BOLF and add the BOLF to an appropriately labeled 15 milliliter conical tube on ice. To isolate the exosomes, centrifuge the BOLF samples and transfer the supernatants to new 15 milliliter tubes.Centrifuge the samples again and transfer the supernatants to ultra-centrifuge tubes to remove the larger, cellular particles. Transfer the supernatants to new conical tubes and load the samples into 0.2 micron syringe filters to remove any remaining larger particles. Add the filtered samples to new ultra-centrifuge tubes to pellet the exosomes.Carefully discard the supernatants and re-suspend the pellets in 100 microliters of PBS per tube. Then transfer the exosome samples into individual 1.5 milliliter tubes for their minus 80 degree Celsius storage. To purify the exosomes, add 0.4 microliters of the appropriate exosome dye to one tube containing 100 microliters of suspension solution per sample and quickly add one thawed exosome sample to each tube of dye.Thoroughly mix the exosomes with the dye and incubate the samples at room temperature for five minutes, protected from light. At the end of the incubation, place one exosome spin column per sample into a 1.5 milliliter illusion tube and transfer the exosome solutions to the center of each spin column. Then centrifuge the samples, discard the columns, and store the alluded samples at minus 20 degrees Celsius.To measure exosome uptake by recipient cells, first seed one times 10 to the fourth mouse lung epithelial 12 cells in 0.5 milliliters of medium into each well of an eight well chamber slide for a 16 hour culture at 37 degrees Celsius in 5%carbon dioxide. After thawing, add 10 microliters of the purified fluorescent labeled exosomes to each well and return the cell cultures to the incubator for one hour. At the end of the incubation, wash the cells with PBS and fix each co-culture with 1%paraformaldehyde for 10 minutes at room temperature.Remove the medium and then mount the slide with medium supplemented with DAPI and image the samples by fluorescence microscopy at a 40 times magnification. To verify exosome interaction with recipient cells, seed two times 10 to the fifth mouse lung epithelial 12 cells in two milliliters of medium per well in a 12 well culture plate containing the appropriate culture medium for a two hour incubation at 37 degrees Celsius and 5%carbon dioxide. At the end of the incubation, add 20 micrograms of exosomes to three wells of epithelial cells per experimental group and return the cells to the incubator.After 24 hours, remove the medium, wash the cells with PBS, and add 600 microliters of lysis buffer to each well with gentle shaking. Then transfer the entire contents of each well into individual 1.5 milliliter tubes and analyze the samples according to the standard real time PCR protocols. Within 24 hours of LPS administration, neutrophilic influx is observed in the lungs of LPS treated animals.After their isolation and purification as just demonstrated, the presence of exosomes in the BOLF from septic lung injury animals can be confirmed by transmission electron microscopy. The expression of select pro-inflammatory micro RNA is significantly increased in bronchoalveolar fluid exosomes from LPS treated animals compared to controlled PBS treated mice. LPS treated exosomes are taken up by mouse bronchiole epithelial cells as evidenced by the co-localization of fluorescent dye labeled exosomes with DAPI labeled cells after one hour of co-culture.Pro-inflammatory mediator expression is significantly increased in epithelial cells co-cultured with exosomes from LPS treated animals compared to co-culture with exosomes from control PBS treated mice. Further, cells treated with exosomes from LPS treated mice demonstrate a significantly attenuated expression of zonula occludens one compared to controls indicating a less intact cell integrity after LPS treated exosome co-culture. The intra-peritoneal injection of LPS, the bronchoalveolar lavage extraction, and exosome delegation from the bronchoalveolar lavage all the steps can be completed in a relatively short amount of time.Following this procedure, other methods like exosome and epithelial cell co-culture can be performed to answer additional questions about the cross talk between innate and structural cells in the lung alveolar micro-environment. Cross talk between innate immune and structural cells via exosome shuttling contributes to the inflammatory response and to structural barrier disruption suggesting exosomal micro-RNA targeting may provide a normal platform for treating acute lung injury and acute respiratory distress syndrome. After watching this video you should have a good understanding of how to induce septic lung injury in mice, isolate bronchoalveolar lavage fluid from mice, isolate exosomes from the lavage fluid, and use a co-culture model for interrogating intracellular cross talk.Don't forget that working with LPS can be hazardous and that precautions such as wearing the appropriate personal protective equipment should always be taken while performing this procedure.

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Research

JoVE Journal

Genetics

siRNA Transfection and EMSA Analyses on Freshly Isolated Human Villous Cytotrophoblasts

Human primary villous cytotrophoblasts are a very useful source of primary cells to study placental functions and regulatory mechanisms, and to comprehend diseases related to pregnancy. In this protocol, human primary villous cytotrophoblasts freshly isolated from placentas through a standard DNase/trypsin protocol are microporated with small interfering RNA (siRNA). This approach provided greater efficiency for siRNA transfection when compared to a lipofection-based method. Transfected cells can subsequently be analyzed by standard Western blot within a time frame of 3-4 days post-transfection. In addition, using cultured primary villous cytotrophoblasts, Electrophoretic Mobility Shift Assay (EMSA) analysis was optimized and performed on extracts from days 1 to 4. The use of these cultured primary cells and the protocol described allow for an evaluation of the implication of specific genes and transcription factors in the process of villous cytotrophoblast differentiation into a syncytiotrophoblast-like cell layer. However, the limited time span allowable in culture precludes the use of methods requiring more time, such as generation of a stable cell population. Therefore testing of this cell population requires highly optimized gene transfer protocols.

This protocol describes a method for efficiently transfecting siRNA in freshly isolated human villous cytotrophoblasts using microporation and identifying DNA-protein complexes in these cells. Transfected cells can be monitored by Western blot and EMSA analyses during the 4-day culture time.

Human primary villous cytotrophoblasts are a very useful source of primary cells to study placental functions and regulatory mechanisms, and to comprehend ...

In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining

The mitochondrial genome is inherited exclusively through the maternal line. Understanding of how the mitochondrion and its genome are proliferated and transmitted from one generation to the next through the female oocyte is of fundamental importance. Because of the genetic tractability, and the elegant, ordered simplicity by which oocyte development proceeds, Drosophila oogenesis has become an invaluable system for mitochondrial study. An EdU (5-ethynyl-2&#180;-deoxyuridine) labeling method was utilized to detect mitochondrial DNA (mtDNA) replication in Drosophila ovaries. This method is superior to the BrdU (5-bromo-2'-deoxyuridine) labeling method in that it allows for good structural preservation and efficient fluorescent dye penetration of whole-mount tissues.
Here we describe a detailed protocol for labeling replicating mitochondrial DNA in Drosophila adult ovaries with EdU. Some technical solutions are offered to improve the viability of the ovaries, maintain their health during preparation, and ensure high-quality imaging. Visualization of newly synthesized mtDNA in the ovaries not only reveals the striking temporal and spatial pattern of mtDNA replication through oogenesis, but also allows for simple quantification of mtDNA replication under various genetic and pharmacological perturbations.

In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining

Drosophila oogenesis continues to be exceptionally useful in the study of mitochondrial proliferation and inheritance. This manuscript describes a detailed protocol used to label the replicating mitochondrial DNA (mtDNA) in Drosophila adult ovaries with 5-ethynyl-2&#180;-deoxyuridine (EdU), which facilitates uncovering mechanisms associated with mitochondrial inheritance that were previously debatable.

The mitochondrial genome is inherited exclusively through the maternal line. Understanding of how the mitochondrion and its genome are proliferated and ...

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most mutagenesis-based screens, researchers have relied on the use of toxic chemicals, carcinogens, or irradiation, which requires designated equipment, safety setup, and/or disposal of hazardous materials. We have developed a simple approach to induce heritable mutations in C. elegans using germline-expressed histone-miniSOG, a light-inducible potent generator of reactive oxygen species. This mutagenesis method is free of toxic chemicals and requires minimal laboratory safety and waste management. The induced DNA modifications include single-nucleotide changes and small deletions, and complement those caused by classical chemical mutagenesis. This methodology can also be used to induce integration of extrachromosomal transgenes. Here, we provide the details of the LED setup and protocols for standard mutagenesis and transgene integration.

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Genetically-encoded histone-miniSOG induces genome-wide heritable mutations in a blue&#160;light-dependent manner. This mutagenesis method is simple, fast, free of toxic chemicals, and well-suited for forward genetic screening and transgene integration.

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most ...

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative without tools to study gene function. The development and sharing of detailed protocols for the establishment of new model systems in laboratories, and for tools to carry out functional studies, is thus crucial for leveraging the power of genomics. Coleoptera (beetles) are the largest clade of insects and occupy virtually all types of habitats on the planet. In addition to providing ideal models for fundamental research, studies of beetles can have impacts on pest control as they are often pests of households, agriculture, and food industries. Detailed protocols for rearing and maintenance of D. maculatus laboratory colonies and for carrying out dsRNA-mediated interference in D. maculatus are presented. Both embryonic and parental RNAi procedures-including apparatus set up, preparation, injection, and post-injection recovery-are described. Methods are also presented for analyzing embryonic phenotypes, including viability, patterning defects in hatched larvae, and cuticle preparations for unhatched larvae. These assays, together with in situ hybridization and immunostaining for molecular markers, make D. maculatus an accessible model system for basic and applied research. They further provide useful information for establishing procedures in other emerging insect model systems.

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Here, we present protocols for rearing an intermediate-germ beetle, Dermestes maculatus (D. maculatus) in the lab. We also share protocols for embryonic and parental RNAi and methods for analyzing embryonic phenotypes to study gene function in this species.

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative ...

Pooled shRNA Screen for Reactivation of MeCP2 on the Inactive X Chromosome

Forward genetic screens using reporter genes inserted into the heterochromatin have been extensively used to investigate mechanisms of epigenetic control in model organisms. Technologies including short hairpin RNAs (shRNAs) and clustered regularly interspaced short palindromic repeats (CRISPR) have enabled such screens in diploid mammalian cells. Here we describe a large-scale shRNA screen for regulators of X-chromosome inactivation (XCI), using a murine cell line with firefly luciferase and hygromycin resistance genes knocked in at the C-terminus of the methyl CpG binding protein 2 (MeCP2) gene on the inactive X-chromosome (Xi). Reactivation of the construct in the reporter cell line conferred survival advantage under hygromycin B selection, enabling us to screen a large shRNA library and identify hairpins that reactivated the reporter by measuring their post-selection enrichment using next-generation sequencing. The enriched hairpins were then individually validated by testing their ability to activate the luciferase reporter on Xi.

We report a small hairpin RNA (shRNA) and next generation sequencing-based protocol for identifying regulators of X-chromosome inactivation in a murine cell line with firefly luciferase and hygromycin resistance genes fused to the methyl CpG binding protein 2 (MeCP2) gene on the inactive X chromosome.

Forward genetic screens using reporter genes inserted into the heterochromatin have been extensively used to investigate mechanisms of epigenetic control ...

Formaldehyde-assisted Isolation of Regulatory Elements to Measure Chromatin Accessibility in Mammalian Cells

Appropriate gene expression in response to extracellular cues, that is, tissue- and lineage-specific gene transcription, critically depends on highly defined states of chromatin organization. The dynamic architecture of the nucleus is controlled by multiple mechanisms and shapes the transcriptional output programs. It is, therefore, important to determine locus-specific chromatin accessibility in a reliable fashion that is preferably independent from antibodies, which can be a potentially confounding source of experimental variability. Chromatin accessibility can be measured by various methods, including the Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) assay, that allow the determination of general chromatin accessibility in a relatively low number of cells. Here we describe a FAIRE protocol that allows simple, reliable, and fast identification of genomic regions with a low protein occupancy. In this method, the DNA is covalently bound to the chromatin proteins using formaldehyde as a crosslinking agent and sheared to small pieces. The free DNA is afterwards enriched using phenol:chloroform extraction. The ratio of free DNA is determined by quantitative polymerase chain reaction (qPCR) or DNA sequencing (DNA-seq) compared to a control sample representing total DNA. The regions with a looser chromatin structure are enriched in the free DNA sample, thus allowing the identification of genomic regions with lower chromatin compaction.

The plasticity of nuclear architecture is controlled by dynamic epigenetic mechanisms including non-coding RNAs, DNA methylation, nucleosome repositioning as well as histone composition and modification. Here we describe a Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) protocol which allows the determination of chromatin accessibility in a reproducible, inexpensive, and straightforward manner.

Appropriate gene expression in response to extracellular cues, that is, tissue- and lineage-specific gene transcription, critically depends on highly ...

CRISPR-Mediated Reorganization of Chromatin Loop Structure

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene expression, but whether looping is responsible for or a result of alterations in gene expression is still unknown. Until recently, how chromatin looping affects the regulation of gene activity and cellular function has been relatively ambiguous, and limitations in existing methods to manipulate these structures prevented in-depth exploration of these interactions. To resolve this uncertainty, we engineered a method for selective and reversible chromatin loop re-organization using CRISPR-dCas9 (CLOuD9). The dynamism of the CLOuD9 system has been demonstrated by successful localization of CLOuD9 constructs to target genomic loci to modulate local chromatin conformation. Importantly, the ability to reverse the induced contact and restore the endogenous chromatin conformation has also been confirmed. Modulation of gene expression with this method establishes the capacity to regulate cellular gene expression and underscores the great potential for applications of this technology in creating stable de novo chromatin loops that markedly affect gene expression in the contexts of cancer and development.

Chromatin looping plays a significant role in gene regulation; however, there have been no technological advances that allow for selective and reversible modification of chromatin loops. Here we describe a powerful system for chromatin loop re-organization using CRISPR-dCas9 (CLOuD9), demonstrated to selectively and reversibly modulate gene expression at targeted loci.

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene ...

Identification of Homologous Recombination Events in Mouse Embryonic Stem Cells Using Southern Blotting and Polymerase Chain Reaction

Relative to the issues of off-target effects and the difficulty of inserting a long DNA fragment in the application of designer nucleases for genome editing, embryonic stem (ES) cell-based gene-targeting technology does not have these shortcomings and is widely used to modify animal/mouse genome ranging from large deletions/insertions to single nucleotide substitutions. Notably, identifying the relatively few homologous recombination (HR) events necessary to obtain desired ES clones is a key step, which demands accurate and reliable methods. Southern blotting and/or conventional PCR are often utilized for this purpose. Here, we describe the detailed procedures of using those two methods to identify HR events that occurred in mouse ES cells in which the endogenous Myh9 gene is intended to be disrupted and replaced by cDNAs encoding other nonmuscle myosin heavy chain IIs (NMHC IIs). The whole procedure of Southern blotting includes the construction of targeting vector(s), electroporation, drug selection, the expansion and storage of ES cells/clones, the preparation, digestion, and blotting of genomic DNA (gDNA), the hybridization and washing of probe(s), and a final step of autoradiography on the X-ray films. PCR can be performed directly with prepared and diluted gDNA. To obtain ideal results, the probes and restriction enzyme (RE) cutting sites for Southern blotting and the primers for PCR should be carefully planned. Though the execution of Southern blotting is time-consuming and labor-intensive and PCR results have false positives, the correct identification by Southern blotting and the rapid screening by PCR allow the sole or combined application of these methods described in this paper to be widely used and consulted by most labs in the identification of genotypes of ES cells and genetically modified animals.

Here, we present a detailed protocol for identifying homologous recombination events that occurred in mouse embryonic stem cells using Southern blotting and/or PCR. This method is exemplified by the generation of nonmuscle myosin II genetic replacement mouse models using traditional embryonic stem cell-based homologous recombination-mediated targeting technology.

Relative to the issues of off-target effects and the difficulty of inserting a long DNA fragment in the application of designer nucleases for genome ...

Characterizing Histone Post-translational Modification Alterations in Yeast Neurodegenerative Proteinopathy Models

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives each year. Effective treatment options able to halt disease progression are lacking. Despite the extensive sequencing efforts in large patient populations, the majority of ALS and PD cases remain unexplained by genetic mutations alone. Epigenetics mechanisms, such as the post-translational modification of histone proteins, may be involved in neurodegenerative disease etiology and progression and lead to new targets for pharmaceutical intervention. Mammalian in vivo and in vitro models of ALS and PD are costly and often require prolonged and laborious experimental protocols. Here, we outline a practical, fast, and cost-effective approach to determining genome-wide alterations in histone modification levels using Saccharomyces cerevisiae as a model system. This protocol allows for comprehensive investigations into epigenetic changes connected to neurodegenerative proteinopathies that corroborate previous findings in different model systems while significantly expanding our knowledge of the neurodegenerative disease epigenome.

This protocol outlines experimental procedures to characterize genome-wide changes in the levels of histone post-translational modifications (PTM) occurring in connection with the overexpression of proteins associated with ALS and Parkinson&#39;s disease in Saccharomyces cerevisiae models. After SDS-PAGE separation, individual histone PTM levels are detected with modification-specific antibodies via Western blotting.

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives ...

Determining the Egg Fertilization Rate of Bemisia tabaci Using a Cytogenetic Technique

A few species of sap-sucking whiteflies are some of the most damaging terrestrial pests worldwide because of the crop damage they inflict and plant viruses they vector. Despite numerous studies of the biology of these species in different environments, a key life history parameter, offspring sex ratios, has received little attention, yet is important for predicting population dynamics. The primary sex ratio (sex ratio at oviposition) of Bemisia tabaci has never been reported but can be found by determining the egg fertilization rate of this haplodiploid insect. The technique involves the dechorionation of eggs with bleach, a series of fixation steps, and the application of the general DNA fluorescent stain, DAPI (4&#8242;,6-diamidino-2-phenylindole, a DNA-binding fluorescent dye), to bind to female and male pronuclei. Here, we present the technique, and an example of its application, to test whether an endosymbiotic bacterium, Rickettsia sp. nr. bellii, influenced the primary sex ratio of B. tabaci. This method may assist in population studies of whiteflies, or in determining if sex allocation exists with certain environmental stimuli.

Determining the Egg Fertilization Rate of Bemisia tabaci Using a Cytogenetic Technique

We present a simple cytogenetic technique using 4&#8242;,6-diamidino-2-phenylindole (DAPI) to determine the fertilization rate and primary sex ratio of the haplodiploid invasive pest Bemisia tabaci.

A few species of sap-sucking whiteflies are some of the most damaging terrestrial pests worldwide because of the crop damage they inflict and plant ...